Significant improvements in the mariculture of abalone and other gastropod molluscs are presented in U.S. Pat. Nos. 4,183,322, 4,226,210, and 4,253,418, the disclosures of which are incorporated herein by reference. The present invention is directed to further advances in growing large numbers of healthy abalone through early life stages when some bacteria and other micro-organisms are potentially lethal, and at least one bacterium is essential.
We here disclose processes for the prespawning preparation of adult abalone, and improved processes for egg cleaning, hatching, larval growing, settlement, and post-settlement life, whereby substantially greater numbers of animals survive the first two months of life when high mortality is otherwise experienced. These improved processes include the use of bacteria-free water at each stage; the use of separately cultured selected bacteria for inducing settlement, providing early nutrition, and maintaining water quality control; and the use of antibiotics to control the growth of unwanted bacteria. A further refinement involves addition of nutrients to the water bath for inducing growth of the desired bacteria and improving the effectiveness of the antibiotics being used.
U.S. Pat. No. 4,183,322 discloses the process of cleaning recently spawned abalone eggs, hatching them, and growing the larvae. It describes the spawning and fertilization process, and teaches the following steps to obtain a high level of hatching and larvae survival:
1. Separation of eggs from the body of seawater into which they are spawned through a multiple screening process using seawater that has been filtered and treated to reduce the bacterial count to less than about 100 cells per milliliter. PA1 2. Hatching the eggs in water of a bacterial count of 100 cells per milliliter or less. PA1 3. Growing the hatched larvae in seawater filtered to remove large components, but coarse enough (passing particles of about 25 microns or smaller) to retain biological components that control ammonia. PA1 4. Maintaining temperature control, dissolved oxygen levels, and approximate egg and larval densities for various container configurations. PA1 1. Preconditioning of settlement surfaces to induce settlement and provide early nutrition by providing thereon a microbial community. PA1 2. Using seawater free of, or with a minimum of, components harmful to the young abalone. PA1 3. Maintaining satisfactory water quality by controlling the biological balance through induced photosynthesis with certain strains of algae and the addition of plant nutrients to provide food and to control waste products. PA1 4. Control of temperature, oxygen levels, larval densities, water circulation, and other factors. PA1 (1) use of seawater that is free of harmful components, including obtaining this quality of seawater by prefiltration; and PA1 (2) conditioning the settlement surfaces by using separately cultured strains of bacteria or other microorganisms. PA1 (1) They can use single carbon compounds for energy and growth, and ammonia as a sole source of nitrogen. PA1 (2) They do not require vitamins or other growth factors. PA1 (3) They carry out a respiratory metabolism as opposed to a fermentative metabolism. PA1 (4) They do not produce toxic metabolic products or release more complex toxic materials. PA1 (1) It is a Gram-negative rod, motile with polar multitrichous flagellation. PA1 (2) Its metabolism is respiratory rather than fermentative, and it cannot reduce nitrate to nitrite, nor live anaerobically by denification. PA1 (3) It does not fix nitrogen, is catalase-positive, gives a weak to negative oxidase reaction, and does not produce indole. PA1 (4) It stores poly-.beta.-hydroxybutyrate as a cellular reserve. PA1 (5) It produces a yellow non-florescent pigment on some media. PA1 (6) It does not produce arginine dihydrolase, nor are extracellular hydrolases for gelatin or starch produced. PA1 (7) It has no special growth factor requirements and can grow with ammonia or nitrate as a source of nitrogen. PA1 (8) It can use any of the following compounds as a sole source of carbon and energy: D-ribose, D-glucose, D-mannose, D-galactose, D-fructose, sucrose, trehalose, maltose, gluconate, acetate, propionate, butyrate, malonate, succinate, DL-malate, DL-.beta.-hydroxybutyrate, DL-lactate, citrate, pyruvate, mannitol, meso-inositol, glycerol, ethanol, n-propanol, glycine, L-.alpha.-alanine, .beta.-alanine, L-serine, L-leucine, L-isoleucine, L-aspartate, L-glutamate, L-lysine, L-arginine, L-ornithine, L-tyrosine, .lambda.-aminobutyrate, L-proline, L-phenylalanine, betaine, and asparagine. PA1 (9) It cannot use any of the following compounds as a sole source of carbon and energy: D-xylose, D-arabinose, L-arabinose, D-fucose, L-rhamnose, cellobiose, lactose, starch, 2-ketogluconate, caproate, pelargonate, oxalate, maleate, adipate, DL-tartrate, glycollate, laevulinate, sorbitol, ethylene glycol, dulcitol, methanol, isopropanol, n-butanol, geraniol, benzoate, p-hydroxybenzoate, m-hydroxybenzoate, phenylacetate, phenol, testosterone, L-threonine, DL-norleucine, L-valine, .delta.-aminovalerate, L-citruline, L-histidine, L-tryptophan, p-aminobenzoate, putrescine, histamine, creatine, pantothenate, acetamide, and nicotinate. PA1 a. Maintaining previously cleaned (brush scrubbed) adult animals in essentially bacteria-free seawater containing one or more antibiotics in solution for a period of at least 48 hours prior to spawning, or for a period sufficient to reduce the level of deleterious marine bacteria associated with the adult animals. PA1 b. Replacing the antibiotic-containing seawater with essentially bacteria-free seawater. The spawning and fertilization processes, and the separation of the fertilized eggs, use techniques described in U.S. Pat. No. 4,183,322. PA1 c. Placing the separated, cleaned, and fertilized eggs in a second body of essentially bacteria-free seawater containing one or more antibiotics for a period necessary for incubation and hatching. PA1 d. Selectively transferring the resultant larvae substantially free of hatching debris by the process described in U.S. Pat. No. 4,183,322, to a third body of essentially bacteria-free seawater containing one or more antibiotics, and adding antibiotic-resistant strains of beneficial bacteria to this seawater bath. PA1 e. Transferring the developed larvae from the third body of seawater into a settling tank (as described in U.S. Pat. No. 4,253,418) of essentially bacteria-free seawater, into which is added one or more antibiotics, and selected and separately cultured strains of beneficial antibiotic-resistant bacteria (as described above) that will induce settlement of the free-swimming larvae, provide food for the developing abalone after settlement and metamorphosis, and metabolize ammonia released by the larvae through their metabolic processes. PA1 a. The use of one antibiotic compound in any one or more of the above steps. PA1 b. The use of a combination of one or more of the antibiotics in one or more of the above steps. PA1 c. The use of a sequence of antibiotics through two or more stages.
This process significantly reduces early mortality in that 90 percent of the eggs hatch, and 90 percent of very young larvae survive.
We have found, however, that the high survival rates experienced at this very early stage of life are not maintained during later stages of life. The increase in mortality rate is believed due to pathogenic wild bacteria, introduced from the parent abalone in the hatching bath and in the larval-growing bath, which infect the animals at this early stage.
U.S. Pat. No. 4,226,210 describes the biological process of larval settlement and metamorphosis, and the early nutritional stages of abalone and other benthic gastropod molluscs. The patent also discloses that, during the approximately first 60 days of the young abalone's crawling life, there is high mortality due to various causes, including bacteria, the quality of the settlement surface, and the nature of the microbial community thereon. Although the process therein described provides for substantial control over these deleterious effects with major improvements in survival over nature, this patent also recognizes that substantial mortality still occurs.
This patent also teaches that economical quantities of abalone can be settled and grown using method steps including the following:
One preferred practice disclosed in U.S. Pat. No. 4,226,210 is to filter coastal seawater to remove large components, while retaining smaller wild microorganisms smaller than 5 to 10 microns in size, and then, for a period of several days, flowing such water through the settlement tank and over the settlement surfaces for formation of a settlement-inducing microbial community. This patent also suggests:
The techniques taught by this patent have resulted in significant improvements in mariculture production, but even greater reduction of animal mortality is desired.
The purpose of the present invention is to advance the art of culturing abalone through the egg, larval, and post-larval early life stages by precisely controlling all biological processes to prevent the entry and growth of harmful bacteria and other harmful biological components, while providing, through controlled cultured processes, sufficient quantities and qualities of bacteria or other microorganisms to induce a high level of settlement, and to provide adequate early nutrition for high survival and rapid growth through the first several months of life.
Both of these above-discussed patents describe the harmful effects of bacteria during the egg, larval, and post-larval stages, and U.S. Pat. No. 4,183,322 provides for the use of wild components of a bacterial size to control ammonia. Similarly, U.S. Pat. No. 4,226,210 provides for the use of wild bacteria and other similarly sized biological components for preconditioning settlement surfaces to induce settlement and provide early nutrition. Counts of wild bacteria approximating 100 cells per milliliter are allowed which, when used to fill the 2-million milliliter tanks routinely used in production operations for egg hatching, or for larval growing, or for settlement, as described in U.S. Pat. No. 4,253,418, allows up to 2.times.10.sup.8 total bacteria to be introduced in each step. It is now believed that the introduction of such large numbers of microorganisms permits the introduction of harmful pathogens which can cause excessive mortality in later stages of life.
We have found that the use of bacteria-free seawater, in combination with the use of antibiotics and selected strains of separately cultured bacteria, greatly improves survival rates for young abalone through the first 60 days of life. The processes of this invention substantially reduce the probability of introducing harmful organisms, provide separately cultured needed bacteria, and provide antibiotics to control unwanted pathogens that are accidentally introduced.
It is known that young abalone need bacteria in their environment for consuming ammonia during the larval-rearing stages. The practice of this invention first involves isolation from the wild of superior strains of bacteria that consume ammonia during the larval stages, induce settlement in metamorphosis of larvae, and provide nutrition during the immediate post-settlement period. This process involves isolating from unfiltered, natural seawater different bacterial strains by using standard laboratory procedures for the isolation and culturing of marine bacteria.
The next step involves testing each isolated pure culture of these bacteria. Broth cultures, incubated for 24 to 48 hours, are diluted with 0.2 micron membrane-filtered seawater and added to sterile dishes containing 100 milliliters of the same filter-sterilized water. Fifty to one hundred swimming larvae are added to each of five replicate bowls for each dilution of culture tested. After addition of the larvae and bacteria, the bowls are placed in a controlled-temperature environment at a suitable temperature for that particular abalone species, and monitored each day for the number of larvae that have settled, the number that are dead, and the number remaining in the free-swimming state.
After an approximately 30-day period, such a screening process will identify some bacterial strains that are pathogenic, some that have no effect, and some that induce settlement and support rapid growth during the subsequent post-settlement period. The superior strains are then maintained in culture using standard bacteriological laboratory techniques. The selective isolation, testing, culturing, and utilization of such favorable bacterial strains is a important facet of this invention.
Bacteria that we have found to have the desired beneficial effect have the following properties in common:
The bacterium of choice most closely resembles Pseudomonas cepacia and Pseudomonas marginata, and is described in greater detail below.
It is a further major feature of this invention to select and culture strains of the preferred bacteria that grow and thrive in the presence of the preferred antibiotics. Such resistant strains are useful in providing water quality control, settlement inducement, and nutrition necessary to sustain early life at the same time that a compatible antiobiotic compound is used to control harmful bacteria.
Another aspect of this invention is to select and culture strains of the preferred bacteria that are resistant to bacteriophages, which are bacteria-degrading, filterable, and naturally occuring viruses that can kill the preferred population of select bacteria in a very short time. Yet another feature is the addition of chemical compounds which provide nutrition for the preferred bacteria, and which, because of their growth-stimulating qualities, make the antibiotic compounds more effective by increasing the susceptibility of unwanted bacteria through growth stimulation. The practice of these features of this invention will be further described below.
In making the advancements herein disclosed it was found that elimination or control of harmful pathogenic bacteria during the first two months of abalone life would eliminate a major cause of mortality. When using coastal seawater in prior art, counts of wild bacteria can be as high as 10,000 cells per cubic centimeter (milliliter) for the unfiltered seawater. Even after the filtration and sterilization process described in the prior-art patents, the number of pathogenic strains, although a small percentage of the total wild population, can be sufficiently significant in size to infect a culture of young abalone and cause massive mortality sometime during the next 60-day period. Therefore, processes for eliminating wild bacteria entirely, or almost entirely, were developed.
Although we have now found the ideal is to eliminate all wild bacteria entirely from the culturing process in order to eliminate infectious pathogens, this ideal is virtually impossible in production. While seawater can be treated to eliminate all bacteria, and although tanks, utensils, and equipment can be sterilized by heat, chemicals, and other means, the abalone eggs carry bacteria obtained from the mother at the time of spawning. At the present time, no process exists for obtaining eggs in a bacteria-free condition from the mother abalone. Therefore, pathogens or potential pathogens are always present, and must be either destroyed or controlled in such a manner as to prevent development of sufficient populations to cause significant mortality in the post-larval abalone.
In the course of developing these processes, it was discovered that the principal pathogens were marine gram-negative bacteria. It was further discovered that one or more antibiotic compounds of the group consisting of polymyxin B, chloramphenicol, ampicillin, erythromycin, and chlortetracycline, were highly effective in either eliminating or controlling the growth of such pathogenic bacteria. Other antibiotics, such as neomycin, streptomycin, and gentamicin, are also effective, but we prefer to use one or more of those compounds listed above in the practice of this invention. While these antibiotic compounds are our preferred practice, this invention is not limited to using only those compounds listed.